Experimental Models of In Vitro Blood-Brain Barrier for CNS Drug Delivery: An Evolutionary Perspective.

Central nervous system (CNS) disorders represent one of the leading causes of global health burden. Nonetheless, new therapies approved against these disorders are among the lowest compared to their counterparts. The absence of reliable and efficient in vitro blood-brain barrier (BBB) models resembling in vivo barrier properties stands out as a significant roadblock in developing successful therapy for CNS disorders. Therefore, advancement in the creation of robust and sensitive in vitro BBB models for drug screening might allow us to expedite neurological drug development. This review discusses the major in vitro BBB models developed as of now for exploring the barrier properties of the cerebral vasculature. Our main focus is describing existing in vitro models, including the 2D transwell models covering both single-layer and co-culture models, 3D organoid models, and microfluidic models with their construction, permeability measurement, applications, and limitations. Although microfluidic models are better at recapitulating the in vivo properties of BBB than other models, significant gaps still exist for their use in predicting the performance of neurotherapeutics. However, this comprehensive account of in vitro BBB models can be useful for researchers to create improved models in the future.

Nanoparticle-Induced Controlled Drug Delivery Using Chitosan-Based Hydrogel and Scaffold: Application to Bone Regeneration.

The novel chitosan nanohybrid hydrogel and scaffold have been developed with high mechanical strength and tailor the drug release ability for their applications in the biomedical arena. Nanohybrid hydrogels are prepared in dilute acetic acid medium using two different types of two-dimensional-layered nanoparticles. Scaffolds are prepared through lyophilization of hydrogels. Highly porous, open, and 3D interconnected morphologies are observed in the nanohybrid scaffolds, as opposed to the thick wall, smaller pore dimension in pure chitosan. The interaction between the nanoparticles and chitosan chains are elucidated using different spectroscopic techniques, which in turn are responsible for the uniform distribution of the nanoparticle in the chitosan matrix. Nanohybrids are found to be highly mechanically stable in both states (hydrogel and scaffold), as compared to pure chitosan because of the good reinforcing ability of 2D nanoparticles. Sustained drug release has been achieved in nanohybrid in vitro, as compared to the pure chitosan hydrogel/scaffold, mainly due to greater interactions between the components and the better barrier effect of 2D nanoparticles. Cytotoxicity of the nanohybrids is verified using NIH 3T3 mouse embryonic fibroblast cells for their possible use as controlled drug delivery vehicles. Nanohybrids are found to be nontoxic in nature and more biocompatible as compared to pure chitosan, as observed through cell viability and cell imaging studies. Interestingly, cell growth occurs within the pores of the nanohybrid scaffold, vis-à-vis the surface proliferation noticed in the pure chitosan scaffold. Better biocompatibility, hydrophilic nature, and sustained delivery with location specific cell growth make this nanohybrid hydrogel unique for biomedical uses. The bone regeneration rate is found to be significantly higher for the nanohybrid scaffold as compared to blank/pure chitosan without any side effect, suggesting nanohybrid systems are superior biomaterials.

Engineered Cellular Uptake and Controlled Drug Delivery Using Two Dimensional Nanoparticle and Polymer for Cancer Treatment.

Two major problems in chemotherapy, poor bioavailability of hydrophobic anticancer drug and its adverse side effects causing nausea, are taken into account by developing a sustained drug release vehicle along with enhanced bioavailability using two-dimensional layered double hydroxides (LDHs) with appropriate surface charge and its subsequent embedment in polymer matrix. A model hydrophobic anticancer drug, raloxifene hydrochloride (RH), is intercalated into a series of zinc iron LDHs with varying anion charge densities using an ion exchange technique. To achieve significant sustained delivery, drug-intercalated LDH is embedded in poly(ε-caprolactone) (PCL) matrix to develop intravenous administration and to improve the therapeutic index of the drug. The cause of sustained release is visualized from the strong interaction between LDH and drug, as measured through spectroscopic techniques, like X-ray photoelectron spectroscopy, infrared, UV-visible spectroscopy, and thermal measurement (depression of melting temperature and considerable reduction in heat of fusion), using differential scanning calorimeter, followed by delayed diffusion of drug from polymer matrix. Interestingly, polymer nanohybrid exhibits long-term and excellent in vitro antitumor efficacy as opposed to pure drug or drug-intercalated LDH or only drug embedded PCL (conventional drug delivery vehicle) as evident from cell viability and cell adhesion experiments prompting a model depicting greater killing efficiency (cellular uptake) of the delivery vehicle (polymer nanohybrid) controlled by its better cell adhesion as noticed through cellular uptake after tagging of fluorescence rhodamine B separately to drug and LDH. In vivo studies also confirm the sustained release of drug in the bloodstream of albino rats using polymer nanohybrid (novel drug delivery vehicle) along with a healthy liver vis-à-vis burst release using pure drug/drug-intercalated LDHs with considerable damaged liver.

Mannose-Functionalized Chitosan-Coated PLGA Nanoparticles for Brain-Targeted Codelivery of CBD and BDNF for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disease causing cognitive and memory decline. AD is characterized by the deposition of amyloid-ß and hypophosphorylated forms of tau protein. AD brains are found to be associated with neurodegeneration, oxidative stress, and inflammation. Cannabidiol (CBD) shows neuroprotective, antioxidant, and anti-inflammatory properties and simultaneously reduces amyloid-ß production and tau hyperphosphorylation. The brain-derived neurotrophic factor (BDNF) plays a vital role in the development and maintenance of the plasticity of the central nervous system. A decline of BDNF levels in AD patients results in reduced plasticity and neuronal cell death. Current therapeutics against AD are limited to only symptomatic relief, necessitating a therapeutic strategy that reverses cognitive decline. In this scenario, combination therapy of CBD and BDNF could be a fruitful strategy for the treatment of AD. We designed mannose-conjugated chitosan-coated poly(d,l-lactide-co-glycolide (PLGA) (CHTMAN-PLGA) nanoparticles for the codelivery of CBD and BDNF to the brain. Chitosan is modified with mannose to specifically target the glucose transporter-1 (GLUT-1) receptor abundantly present in the blood-brain barrier for selectively delivering therapeutics to the brain. The CBD-encapsulated nanoparticles showed an average hydrodynamic diameter of 306 ± 8.12 nm and a zeta potential of 31.7 ± 1.53 mV. The coated nanoparticles prolonged encapsulated CBD release from the PLGA matrix. The coated nanoparticles exhibited sustained release of CBD for up to 22 days with 91.68 ± 2.91% release of the encapsulated drug. The coated nanoparticles, which had a high positive zeta potential (31.7 ± 1.53 mV), encapsulated the plasmid DNA. The qualitative transfection efficiency was investigated using CHTMAN-PLGA-CBD/pGFP in bEND.3, primary astrocytes, and primary neurons, while the quantitative transfection efficiency of the delivery system was determined using CHTMAN-PLGA-CBD/pBDNF. In vitro, the pBDNF transfection study revealed that the BDNF expression was 4-fold higher for CHTMAN-PLGA-CBD/pBDNF than for naked pBDNF in all of the cell lines. The cytotoxicity and hemocompatibility of the designed nanoparticles were tested in bEND.3 cells and red blood cells, respectively, and the nanoparticles were found to be nontoxic and hemocompatible. Hence, mannose-conjugated chitosan-coated PLGA nanoparticles could be useful as brain-targeting delivery vehicles for the codelivery of CBD and BDNF for possible AD treatment.

Functionalized nanoparticles to deliver nucleic acids to the brain for the treatment of Alzheimer's disease.

Brain-targeted gene delivery across the blood-brain barrier (BBB) is a significant challenge in the 21st century for the healthcare sector, particularly in developing an effective treatment strategy against Alzheimer's disease (AD). The Internal architecture of the brain capillary endothelium restricts bio-actives entry into the brain. Additionally, therapy with nucleic acids faces challenges like vulnerability to degradation by nucleases and potential immune responses. Functionalized nanocarrier-based gene delivery approaches have resulted in safe and effective platforms. These nanoparticles (NPs) have demonstrated efficacy in protecting nucleic acids from degradation, enhancing transport across the BBB, increasing bioavailability, prolonging circulation time, and regulating gene expression of key proteins involved in AD pathology. We provided a detailed review of several nanocarriers and targeting ligands such as cell-penetrating peptides (CPPs), endogenous proteins, and antibodies. The utilization of functionalized NPs extends beyond a singular system, serving as a versatile platform for customization in related neurodegenerative diseases. Only a few numbers of bioactive regimens can go through the BBB. Thus, exploring functionalized NPs for brain-targeted gene delivery is of utmost necessity. Currently, genes are considered high therapeutic potential molecules for altering any disease-causing gene. Through surface modification, nanoparticulate systems can be tailored to address various diseases by replacing the target-specific molecule on their surface. This review article presents several nanoparticulate delivery systems, such as lipid NPs, polymeric micelles, exosomes, and polymeric NPs, for nucleic acids delivery to the brain and the functionalization strategies explored in AD research.

Biocompatible fluorescent probe for detecting mitochondrial alkaline phosphatase activity in live cells.

Alkaline phosphatase (ALP) is an enzyme that actively plays a significant role in the various metabolic processes by transferring a phosphate group to the protein, nucleic acid, etc. The elevated level of ALP in blood plasma is the hallmark of inflammation/cancer. The hyperactive mitochondria in cancer cells produce an excess of ATP to fulfill the high energy demand. Thus, we have developed a fluorescent probe Mito-Phos for ALP, which can detect phosphatase expression in mitochondria in live cells. The probe Mito-Phos has shown ~15-fold fluorescence intensity increments at 450 nm in the presence of 500 ng/mL of ALP. It takes about 60 min to consume the whole amount of ALP (500 ng/mL) in physiological buffer saline. It can selectively react with ALP even in the presence of other probable cellular reactive components. It is highly biocompatible and nontoxic to the live cells. It has shown ALP expression in a dose-dependent manner by providing concomitant fluorescence images in the blue-channel region. It has localized exclusively in the mitochondria in live cells. The probe Mito-Phos is highly biocompatible with the ability to assess ALP expression in mitochondria in live cells.

Injectable Hydrogel through Hydrophobic Grafting on Chitosan for Controlled Drug Delivery.

An injectable hydrogel of chemically modified chitosan has been developed for controlled drug delivery application. Highly hydrophilic chitosan is chemically modified through grafting of ester-diol based polyurethane to transform into hydrogel through hydrophilic-hydrophobic balance. Grafting is confirmed through different spectroscopic techniques such as 13C NMR, UV-visible, and FTIR. Grafted copolymer shows higher contact angle as well as poorer swelling than pure one. Hydrogel of graft copolymer is made in dilute acetic acid medium. Surface morphology of the hydrogel, as investigated through SEM, exhibits an interconnected porous network suitable for drug delivery vehicle. In vitro drug release kinetics reveals that the graft copolymer releases the drug in a sustained manner as compared to the pure one. Two types of model drugs (antibacterial tetracycline hydrochloride and anticancerous doxorubicin hydrochloride) are used for release study to check the control release for various applications. The cytotoxicity of the newly developed hydrogel is accessed using the B16-F10 melanoma cell line, and the hydrogel is found to have a better biocompatible nature than that of pure chitosan. Cellular uptake of drug from graft copolymer hydrogel has considerably increased vis-à-vis pure drug resulting in significant enhancement of cell killing using the developed drug embedded hydrogel system. In vivo gelation study in an animal model verifies that the graft copolymer has the fine ability to be used as an injectable hydrogel for disease control. Hence, the developed graft copolymer has the potential to be used as an injectable hydrogel as a controlled drug delivery vehicle.

Nanohybrid Scaffold of Chitosan and Functionalized Graphene Oxide for Controlled Drug Delivery and Bone Regeneration.

Nanohybrid scaffolds of chitosan have been designed for controlled drug delivery and bone regeneration. Sulfonated graphene oxide has been used to develop the nanohybrids. Nanohybrid scaffolds show highly hydrophilic character and greater mechanical strength as compared to pure chitosan. Nanohybrid scaffolds show an interconnected uniform porous network structure exhibiting sustained release kinetics of the antibacterial drug, tetracycline hydrochloride. Nanohybrids are found to be highly biocompatible in nature and are able to support and proliferate MG63 osteoblast cells and thereby induce bone tissue regeneration. The in-vivo bone healing study shows that the developed nanohybrid scaffolds have the potential to regenerate the bone faster without any side effects as compared to pure scaffolds. Hence, the developed nanohybrid scaffold has good potential as a controlled drug delivery vehicle and in bone tissue engineering for faster healing.

Controlled drug delivery vehicles for cancer treatment and their performance.

Although conventional chemotherapy has been successful to some extent, the main drawbacks of chemotherapy are its poor bioavailability, high-dose requirements, adverse side effects, low therapeutic indices, development of multiple drug resistance, and non-specific targeting. The main aim in the development of drug delivery vehicles is to successfully address these delivery-related problems and carry drugs to the desired sites of therapeutic action while reducing adverse side effects. In this review, we will discuss the different types of materials used as delivery vehicles for chemotherapeutic agents and their structural characteristics that improve the therapeutic efficacy of their drugs and will describe recent scientific advances in the area of chemotherapy, emphasizing challenges in cancer treatments.

Cell proliferation influenced by matrix compliance of gelatin grafted poly(d,l-Lactide) three dimensional scaffolds.

Surface and mechanical properties of the biomaterials are determinants of cellular responses. In our previous study, star-shaped poly(d,l-Lactide)-b-gelatin (ss-pLG) was reported for possessing improved cellular adhesion and proliferation. Here, we extended our investigation to establish the cellular compatibility of gelatin-grafted PDLLA with respect to mechanical properties of biological tissues. In this view, linear PDLLA-b-gelatin (l-pLG) was synthesized and tissue-level compatibility of 1-pLG and ss-pLG against fibroblasts (L929), myoblasts (C2C12) and preosteoblasts (MG-63) was examined. The cell proliferation of C2C12 was significantly higher within l-pLG scaffolds, whereas L929 showed intensified growth within ss-pLG scaffolds. The difference in cell proliferation may be attributed to the varying mechanical properties of scaffolds; where the stiffness of l-pLG scaffolds was notably higher than ss-pLG scaffolds, most likely due to the variable levels of gelatin grafting on the backbone of PDLLA. Therefore, gelatin grafting can be used to modulate mechanical property of the scaffolds and this study reveals the significance of the matrix stiffness to produce the successful 3D scaffolds for tissue engineering applications.